The immune system must be able to discriminate between self and non-self. When self/non-self discrimination fails, the immune system destroys cells and tissues of the body and as a result causes autoimmune diseases. Regulatory T cells actively suppress activation of the immune system and prevent pathological self-reactivity and consequent autoimmune disease. Developing drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune disease is the subject of intense research and, until the development of the present invention, has been largely unsuccessful.
Regulatory T cells (Treg) are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Treg are central to immune system homeostasis, and play a major role in maintaining tolerance to self-antigens and in modulating the immune response to foreign antigens. Multiple autoimmune and inflammatory diseases, including Type 1 Diabetes (T1D), Systemic Lupus Erythematosus (SLE), and Graft-versus-Host Disease (GVHD) have been shown to have a deficiency of Treg cell numbers or Treg function. Consequently, there is great interest in the development of therapies that boost the numbers and/or function of Treg cells.
One treatment approach for autoimmune diseases being investigated is the transplantation of autologous, ex vivo-expanded Treg cells (Tang, Q., et al, 2013. Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in treating animal models of disease and in several early stage human clinical trials, it requires personalized treatment with the patient's own T cells, is invasive, and is technically complex. Another approach is treatment with low dose Interleukin-2 (IL-2). Treg cells characteristically express high constitutive levels of the high affinity IL-2 receptor, IL2Rαβγ, which is composed of the subunits IL2RA (CD25), IL2RB (CD122), and IL2RG (CD132), and Treg cell growth has been shown to be dependent on IL-2 (Malek, T. R., et al., 2010, Immunity, 33:153-65). Clinical trials of low-dose IL-2 treatment of chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis patients (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical trials investigating the efficacy of IL-2 in multiple other autoimmune and inflammatory diseases have been initiated.
Proleukin (marketed by Prometheus Laboratories, San Diego, Calif.), the recombinant form of IL-2 used in these trials, is associated with high toxicity. Proleukin is approved for the treatment of Metastatic Melanoma and Metastatic Renal Cancer, but its side effects are so severe that its use is only recommended in a hospital setting with access to intensive care (Web address: www.proleukin.com/assets/pdf/proleukin.pdf). Until the more recent characterization of Treg cells, IL-2 was considered to be an immune system stimulator, activating T cells and other immune cells to eliminate cancer cells. The clinical trials of IL-2 in autoimmune diseases have employed lower doses of IL-2 in order to target Treg cells, because Treg cells respond to lower concentrations of IL-2 than many other immune cell types due to their expression of IL2Rαβγ (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower doses resulted in safety and tolerability issues, and the treatments used have employed daily subcutaneous injections, either chronically or in intermittent 5 day treatment courses. Therefore, there is a need for an autoimmune disease therapy that potentiates Treg cell numbers and function, that targets Treg cells more specifically than IL-2, that is safer and more tolerable, and that is administered less frequently.
One approach to improving the therapeutic index of IL-2-based therapy is to use variants of IL-2 that are selective for Treg cells relative to other immune cells. IL-2 receptors are expressed on a variety of different immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern likely contributes to its pleiotropic effect on the immune system and high systemic toxicity. The IL-2 receptor exists in three forms: (1) the low affinity receptor, IL2RA, which does not signal; (2) the intermediate affinity receptor (IL2Rβγ), composed of IL2RB and IL2RG, which is broadly expressed on conventional T cells (Tcons), NK cells, eosinophils, and monocytes; and (3) the high affinity receptor (IL2Rαβγ), composed of IL2RA, IL2RB, and IL2RG, which is expressed transiently on activated T cells and constitutively on Treg cells. IL-2 variants have been developed that are selective for IL2Rαβγ relative to IL2Rβγ (Shanafelt, A. B., et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, D. J., et. al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions which reduce their affinity for IL2RB. Because IL-2 has undetectable affinity for IL2RG, these variants consequently have reduced affinity for the IL2Rβγ receptor complex and reduced ability to activate IL2Rβγ-expressing cells, but retain the ability to bind IL2RA and the ability to bind and activate the IL2Rαβγ receptor complex. One of these variants, IL2/N88R (Bay 50-4798), was clinically tested as a low-toxicity version of IL-2 as an immune system stimulator, based on the hypothesis that IL2Rβγ-expressing NK cells are a major contributor to toxicity. Bay 50-4798 was shown to selectively stimulate the proliferation of activated T cells relative to NK cells, and was evaluated in phase I/II clinical trials in cancer patients (Margolin. K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, R. T., et. al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials showed that Bay 50-4798 was considerably safer and more tolerable than Proleukin, and also showed that it increased the levels of CD4+CD25+ T cells, a cell population enriched in Treg cells. Subsequent to these trials, research in the field more fully established the identity of Treg cells and demonstrated that Treg cells selectively express IL2Rαβγ (reviewed in Malek, T. R., et al., 2010, Immunity, 33:153-65). Based on this new research, it can now be understood that IL2Rαβγ selective agonists should be selective for Treg cells.
A second approach to improving the therapeutic index of an IL-2 based therapy is to optimize the pharmacokinetics of the molecule to maximally stimulate Treg cells. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro required a minimum of 5-6 hours exposure to effective concentrations of IL-2 (Cantrell, D. A., et. al., 1984, Science, 224: 1312-1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Because of its short half-life, maintaining circulating IL-2 at or above the level necessary to stimulate T cell proliferation for the necessary duration necessitates high doses that result in peak IL-2 levels significantly above the EC50 for Treg cells or will require frequent administration (FIG. 1). These high IL-2 peak levels can activate IL2Rβγ receptors and have other unintended or adverse effects. An IL-2 analog with a longer circulating half-life than IL-2 can achieve a target drug concentration for a specified period of time at a lower dose than IL-2, and with lower peak levels. Such an IL-2 analog will therefore require either lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of an IL-2 drug will also be more tolerable for patients. A therapeutic with these characteristics will translate clinically into improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy.
One approach to extending the half-life of therapeutic proteins is to conjugate the therapeutic protein with a non-immunogenic water-soluble polymer like polyethylene glycol (PEG). Chemical conjugation of a protein to a PEG molecule (PEGylation) increases the circulating half-life by increasing the effective hydrodynamic radius of the protein, thus reducing the rate at which the protein conjugate is filtered out of blood by the kidney. IL-2 and IL-2 Selective Agonists are relatively small proteins of approximately 15,000 daltons (15 kDa) with a rapid rate of renal clearance. The circulating half-life of PEG molecules increases in proportion to the molecular weight of the PEG (Yamaoka, T., et al., 1994 J Pharm Sci. 83:601-6).
There are a number of factors which impact the successful production and manufacturing of PEGylated therapeutic proteins. First, because PEGylated proteins are prepared by chemical conjugation of a PEG molecule and a protein, it is important that the protein can be manufactured efficiently because of the additional manufacturing steps involved with PEGylation. Second, the PEG moiety should be should be efficiently conjugated to the protein via a specific amino acid residue, leading to a homogeneous product with high yield. Third, the site of PEGylation on the protein should be chosen so as to minimally interfere with the therapeutic activity of the protein. Interference with the protein therapeutic activity could be due to conjugation of the PEG to the active site of the protein, or could be due to steric hinderance of the active site by the PEG. As an example, one form of PEGylated IL-2 was previously reported in which PEG molecules were conjugated to primary amines on IL-2, resulting in a heterogenous mixture of protein species containing between one and four PEG polymers per IL-2 molecule (Katre, N. V. et. al., 1987 Proc Natl Acad Sci USA. 84:1487-91; Knauf, M. J., et. al., 1988 J Biol Chem. 15; 263:15064-701988) which exhibited 4-6 fold reduced biological activity relative to IL-2 (Chen, S. A., 2000 J. Pharmacol Exp Ther. 293:248-59). Because the IL-2 Selective Agonist must bind and activate to a complex of three receptor subunits on Treg cells, the appropriate conjugation site on IL-2 must be chosen carefully for optimal bioactivity.
This invention concerns variants of IL-2 with single amino acid substitutions at specific positions in the IL-2 sequence that enable stable and specific chemical conjugation of PEG molecules while retaining the ability of the IL-2-PEG conjugate to activate Treg cells. These specified amino acid positions, defined as PEG conjugation sites, are chosen such that the IL-2-PEG conjugate resulting from conjugation of a PEG molecule to IL-2 variant is minimally impaired in its ability to bind to and activate the IL2Rαβγ receptor. This invention also concerns IL-2 variants with aforementioned PEG conjugation sites on IL-2 variants that are selective for the IL2Rαβγ receptor, and consequently have high selectivity for Treg cells.
Chemically activated PEGs have been developed with a number of different chemically reactive groups for conjugation to proteins, enabling conjugation to amino acid residues containing primary amines or to thiol groups. Of these, thiol groups, uniquely present on cysteine residues, enable the most selective conjugation of PEGs to proteins, and PEGs with maleimide or iodoacetamide reactive groups react very selectively with free cysteine thiols. Most cysteine residues in extracellular proteins participate in disulfide bonds that stabilize the protein conformation, and the small number of free (unpaired) cysteine residues are usually buried in the interior of proteins (Petersen, M. T., et. al., 1999 Protein Eng. 1999 12:535-48). PEG conjugation to free cysteine residues in a protein requires either a natural exposed free cysteine residue or the introduction of a novel free cysteine residues. The engineering of a novel free cysteine into a protein carries the risk that the introduced novel cysteine can form an inappropriate intrachain disulfide bond with other cysteines, thus causing misfolding of the protein, or can form interchain disulfide bonds with other molecules, thus causing protein aggregation. IL-2 variants with mutated cysteine residues can exhibit substantially reduced activity due to mispaired disulfide bonds (Wang, A., et al., 1984 Science. 224:1431-3). The invention herein focuses on the engineering of novel, free cysteine residues into IL-2 variant proteins that will be compatible with proper protein folding, that enables site-specific conjugation of a thiol-reactive PEG, and results in an IL-2-PEG conjugate that retains the ability to bind to and activate the IL2Rαβγ on Tregs.